The long term objective of this proposal is to understand the molecular mechanism of neurofibrillary degeneration and then based on this knowledge develop therapeutic treatment for Alzheimer disease (AD) and related disorders. Our working hypothesis is (1) that the protein phosphorylation/dephosphorylation is imbalanced in AD brains, leading to abnormal hyperphosphorylation of tau and some other neuronal proteins; (2) that this defect is in part due to a deficit in the phosphoprotein phosphatase (PP) -2A and -1 which leads to the breakdown of microtubules and consequent impairment of axoplasmic flow and retrograde neuronal degeneration; and (3) that inhibition of neurofibrillary degeneration will arrest the progression of AD. Towards this hypothesis we propose to: (1) study the structures and the activities of the two physiological inhibitors of PP-2A, called I1PP2A and I2PP2A from human brain by generation of rabbit affinity purified antibodies to synthetic peptides corresponding to the unique regions of these proteins, isolation of these proteins from brain by column chromatographies and preparative SDS-PAGE, amino acid sequencing of I2PP2A which very much differs in its molecular mass from that in kidney, cloning of these phosphatase inhibitors from a human brain cDNA library, generation and purification of the recombinant brain inhibitors and inhibition of the phosphatase activities by these inhibitors; (2) study the regulation of the activities of PP-2A and PP-1 in AD and age-matched control brains by assaying the levels (by 125I-immuno-dot-blots) and the activities (by radiometric enzyme assays) of I1PP2A, I2PP2A and DARPP-32 in nuclear and cytoplasmic compartments of various areas of these brains, and by immunocytochemical distribution and cellular localization of the inhibitors in various histopathologically affected and unaffected areas of AD and corresponding areas of control brains; and (3) study the effect of the downregulation of PP-2A and PP-1 activities in the phosphorylation of tau and cellular degeneration by generating stably transfected neural progenitor cells isolated and propagated from hippocampus that overexpress the phosphatase inhibitors in a regulatable manner; these studies will include the effect of the overexpression of each phosphatase inhibitor on viability (MTT assay) and morphology of the cells (phase contrast and immunofluorescence), phosphorylation of tau at specific sites with site specific phosphorylation dependent antibodies (125I Western blots), ability of the resulting phosphorylated tau to promote/inhibit microtubule assembly by light scattering assay and sequestration of normal MAPS by binding and sedimentation assays. These studies will help elucidate the role of PP-2A- and PP-1- inhibitors in the abnormal hyperphosphorylation of tau and the neurodegeneration that occurs in AD.